Compounds and compositions for use as modulators of tau aggregation and alleviation of tauopathies

ABSTRACT

This invention relates to the use of bis- and tris-dihydroxyaryl compounds as well as sulfonamides, heteroaryls, tricycloalkyl and their analogs and pharmaceutically acceptable salts, for modulating tau aggregation and alleviating tauopathies, such as Alzheimer&#39;s disease (AD), Pick&#39;s disease (PiD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD) and familial frontotemporal dementia/Parkinsonism linked to chromosome 17 (FTDP-17), amyotrophic lateral sclerosis/Parkinsonism-dementia complex, argyrophilic grain dementia, dementia pugilistic, diffuse neurofibrillary tangles with calcification, progressive subcortical gliosis and tangle only dementia.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 13/010,023filed Jan. 20, 2011 and claims the benefit of priority under 35 USC.§120 to, and is a continuation in part of U.S. application Ser. No.12/269,017 filed Nov. 11, 2008 which is a continuation of U.S.application Ser. No. 10/452,851, filed May 30, 2003 now issued U.S. Pat.No. 7,514,583, which claimed priority under 35 USC §119(e) to:

-   (1) U.S. Provisional Application No. 60/385,144, filed May 31, 2002,-   (2) U.S. Provisional Application No. 60/409,100, filed Sep. 9, 2002,-   (3) U.S. Provisional Application No. 60/412,272, filed Sep. 20,    2002,-   (4) U.S. Provisional Application No. 60/435,880, filed Dec. 20,    2002, and-   (5) U.S. Provisional Application No. 60/463,104, filed Apr. 14,    2003.

This application also claims priority under 35 USC §119(e) to U.S.Provisional Application No. 61/299,005, filed Jan. 28, 2010.

This application claims the benefit of priority under 35 USC §120 to,and is a continuation in part of, U.S. application Ser. No. 12/244,968filed Oct. 3, 2008, which claimed priority under 35 USC §119(e) to U.S.Provisional Application No. 61/001,441, filed Oct. 31, 2007.

The entire contents of all of these applications are incorporated byreference into this application.

TECHNICAL FIELD

This invention relates to the use of bis- and tris-dihydroxyarylcompounds as well as sulfonamides, heteroaryls, tricycloalkyl and theiranalogs and pharmaceutically acceptable esters, and pharmaceuticalcompositions containing them, for modulation of tau aggregation anddissolution/disruption/inhibition of tau aggregates, and alleviation oftauopathies, such as Alzheimer's disease (AD), Pick's disease (PiD),progressive supranuclear palsy (PSP), corticobasal degeneration (CBD)and familial frontotemporal dementia/Parkinsonism linked to chromosome17 (FTDP-17), amyotrophic lateral sclerosis/Parkinsonism-dementiacomplex, argyrophilic grain dementia, dementia pugilistic, diffuseneurofibrillary tangles with calcification, progressive subcorticalgliosis and tangle only dementia.

BACKGROUND OF THE INVENTION

Tau is a microtubule associated protein found primarily in neuronalaxons. Physiological phosphorylation of tau regulates the dynamics ofthe association of tau with tubulin, and thereby microtubule stability(Mazanetz. M. P. and Fischer, P. M. 2007. Nature Reviews 6:464-479). Thestabilization of the microtubules in axons ensures that maintain theirfunction for axonal transport, growth and branching (Bulic, B et al.,2009 Angew. Chem. Int. Ed. 48:2-15). Hyperphosphorylation and misfoldingof the tau protein is thought to be the causative factor in abnormalintracellular aggregation leading ultimately to neuronal dysfunction.Protein aggregates have been found to be toxic to neurons.

Abnormal intraneuronal tau aggregation has three basic pathologicalmanifestations; neurofibrillary tangles (NFT's), neuropil threads (NT's)and the argyrophilic dystrophic neurite plaques (Braak, H and Braak, E,Neurobio. of Aging. 1997 18(4):351-357). Structurally, the NFT's areprincipally comprised of paired helical filaments (PHF) comprised of twofilamentous tau proteins twisted around one another with a crossoverrepeat of 80 nm and a width of 8-20 nm (Li, D., et al., 2008.Computational Biology 4(12) and Kidd, M 1963 Nature, 197:192). There aresix stages (Braak stages I-VI) of tau deposition in the brain, whichprogress temporally at defined anatomical locations with the initialstages characterized primarily by the deposition of NFT's and NT's andthe secondary stages further accompanied by NP (Braak, 1997). In AD andother neuropathies, Braak's stages correlate well with clinical diseaseprogression as demonstrated by increasing cognitive dysfunction. Severecortical destruction which occurs around stages coincides with the firstmanifestations of the clinical onset of AD. Although no tau mutationshave been identified in AD there is a strong correlation between NFTdensity and cognitive decline in AD (Brunden, K. R., Trojanowski, J. Q.,and Lee, V. M. 2009 Nature Reviews 8:783-93).

New biomarkers and models of their temporal characteristics are becomingeven more useful for the diagnosis and characterization of AD (Jack etal., 2010. Lancet 9:119-28). Specifically, tau deposition is associatedwith neurodegeneration in AD and an increase in CSF tau is an importantindicator of tau pathologic changes and correlates well with clinicaldisease severity. A decrease in FDG-PET correlates well with increasedCSF tau and both are valid indicators of synaptic dysfunction (Jack etal, ibid). This model of biomarker ordering, especially in mildlycognitive impaired individuals, has important implications for clinicaltrials. Potential therapeutics could be more accurately assessed forefficacy is they are able to change the trajectory of cognitivedeterioration and individuals might be more selectively chosen fortrials (Jack et al, ibid).

au hyperphosphorylation is a common characteristic of a number ofdementing disorders collectively known as tauopathies, some of whichhave disctinct tau pathology combined with other brain pathologies.Tauopathies include Alzheimer's disease (AD), Pick's disease (PiD),progressive supranuclear palsy (PSP), corticobasal degeneration (CBD)and familial frontotemporal dementia/Parkinsonism linked to chromosome17 (FTDP-17), amyotrophic lateral sclerosis/Parkinsonism-dementiacomplex, argyrophilic grain dementia, dementia pugilistic, diffuseneurofibrillary tangles with calcification, progressive subcorticalgliosis and tangle only dementia. (Spillantini, M G and Goedert M, 1998Trends Neurosci. Oct 21(10):428-33). In AD, tau pathology is typicallylimited to the neurons while other tauopathies can pathologicallyexhibit both neuronal and glial tau deposition (Higuchi, M, et al.,2002. Neuropsychopharmacology: The Fifth Generation of Progress, Chapter94: Tau protein and tauopathy).

It has recently been postulated that tau protein may link Parkinson'sand Alzheimer's disease (Shulman, J. M. and DeJager, P. L. 2009 NatureGenetics 41(12):1261-1262). This study examined whether any genome wideassociation occurs between the two diseases and found that three genesand two new loci were linked to increased susceptibility.

It is presently not known if tau is a causative factor in disease but itis likely that either a loss or gain for function results in pathology.In FTLD17, a missense mutation affects the alternative splicing of tauresulting in the disruption of the ratio of the 4R to 3R tau isoform.More of the 4R isoform with an extra repeat of the microtubule bindingregion may lead to overstabilization of the microtubules resulting indisease. Other post-translational events such as alterations in kinaseactivity and glycosylation could also cause hyperphosphorylation andresult in disease or alternatively proteolytic cleavage could producetruncated tau products more inclined to aggregate (Brunden, ibid).

Recently tau toxicity has been re-emphasized as an important therapeutictarget in neurodegerative tauopathies (Keystone Symposium, March 2009).Routes for developing therapeutics are either directed to inhibitingtau-phosphorylation kinases or seeking compounds effective in themodulation of tau aggregation and/or the dissolution or disruption oftau aggregates which may prove equally useful or more specific for thealleviation of tauopathies (Rafii, M. and Aisen, P. 2009 BMC Medicine7:7). A recent paper surveyed the efficacy of several classes ofcompounds for their ability to prevent tau aggregation and disaggregatepre-formed tau fibrils (Bulic et al.). Although there are generalconcerns regarding the toxicity of disassembled fibrils, Bulic et al.,were able to show that reversing tau aggregation resulted in increasedcell viability.

SUMMARY OF THE INVENTION

In a first aspect, this invention is bis- and tris-dihydroxyarylcompounds and their pharmaceutically acceptable esters, andpharmaceutically acceptable salts thereof for use in the modulation oftau aggregation and dissolution/disruption/inhibition of tau aggregates.

The compounds are:

(1) compounds of the formula:

where:R is a C₁-C₁₀ alkylene group, in which, when the number of carbon atomsis at least 2, there are optionally 1 or 2 non-adjacent double bonds; 1to 3 non-adjacent methylene groups are optionally replaced by NR′ (whereR′ is H, alkyl, or acyl), O, or S; and 1 or 2 methylene groups areoptionally replaced by a carbonyl or hydroxymethylene group; and(2) the compounds that are:3,4,3′,4′-tetrahydroxybenzoin (compound DC-001);3,4,3′,4′-tetrahydroxydesoxybenzoin (compound DC-002);3,4,3′,4′-tetrahydroxydiphenylmethane (compound DC-003);1,2-bis(3,4-dihydroxyphenyl)ethane (compound DC-004);1,3-bis(3,4-dihydroxyphenyl)propane (compound DC-005);3,4,3′,4′-tetrahydroxychalcone (compound DC-006);3,5-bis(3,4-dihydroxyphenyl)-1-methyl-2-pyrazoline (compound DC-007);4,6-bis(3,4-dihydroxyphenyl)-3-cyano-2-methylpyridine (compound DC-008);1,4-bis(3,4-dihydroxybenzyl)piperazine (compound DC-009);N,N′-bis(3,4-dihydroxybenzyl)-N,N′-dimethylethylenediamine (compoundDC-0010); 2,5-bis(3,4-dihydroxybenzyl)-2,5-diaza[2.2.1]bicycloheptane(compound DC-0011);N,N-bis(3,4-dihydroxybenzyl)-trans-1,2-diaminocyclohexane (compoundDC-0012); N,N-bis(3,4-dihydroxybenzyl)-trans-1,4-diaminocyclohexane(compound DC-0013);N,N′-bis(3,4-dihydroxybenzyl)-cis-1,3-bis(aminomethyl)cyclohexane(compound DC-0014); N-(3,4-dihydroxybenzyl)proline3,4-dihydroxybenzylamide (compound DC-0015);2-(3,4-dihydroxybenzyl)isoquinoline-3-carboxylic acid3,4-dihydroxyphenethylamide (compound DC-0016);2,6-bis(3,4-dihydroxybenzyl)-cyclohexanone (compound DC-0017);3,5-bis(3,4-dihydroxybenzyl)-1-methyl-4-piperidinone (compound DC-0018);2,4-bis(3,4-dihydroxybenzyl)-3-tropinone (compound DC-0019);tris-(3,4-dihydroxybenzyl)methane (compound DC-0020);α-(3,4-dihydroxybenzamido)-3,4-dihydroxycinnamic acid3,4-dihydroxybenzyl amide (compound DC-0021);4-(3,4-dihydroxybenzylaminomethylene)-2-(3,4-dihydroxyphenyl)oxazolin-5-one(compound DC-0022); 1,4-bis(3,4-dihydroxybenzoyl)piperazine (compoundDC-0023); N,N′-bis(3,4-dihydroxybenzoyl)-N,N′-dimethylethylenediamine(compound DC-0024);2,5-bis(3,4-dihydroxybenzoyl)-2,5-diaza[2.2.1]bicycloheptane (compoundDC-0025); N,N′-bis(3,4-dihydroxybenzoyl)-trans-1,2-diaminocyclohexane(compound DC-0026);N,N′-bis(3,4-dihydroxybenzoyl)-cis-1,3-bis(aminomethyl)cyclohexane(compound DC-0027); 3,6-bis(3,4-dihydroxybenzyl)-2,5-diketopiperazine(compound DC-0028);3,6-bis(3,4-dihydroxybenzylidene)-1,4-dimethyl-2,5-diketopiperazine(compound DC-0029); N-(3,4-dihydroxyphenylacetyl)proline3,4-dihydroxyanilide (compound DC-0030);2,3-bis(3,4-dihydroxyphenyl)butane (compound DC-0031);1,3-bis(3,4-dihydroxybenzyl)benzene (compound DC-0032);1,4-bis(3,4-dihydroxybenzyl)benzene (compound DC-0033);2,6-bis(3,4-dihydroxybenzyl)pyridine (compound DC-0034);2,5-bis(3,4-dihydroxybenzyl)thiophene (compound DC-0035);2,3-bis(3,4-dihydroxybenzyl)thiophene (compound DC-0036);1,2-bis(3,4-dihydroxyphenyl)cyclohexane (compound DC-0037);1,4-bis(3,4-dihydroxyphenyl)cyclohexane (compound DC-0038);3,7-bis(3,4-dihydroxyphenyl)bicyclo[33.0]octane (compound DC-0039);2,3-bis(3,4-dihydroxyphenyl)-1,7,7-trimethylbicyclo[2.2.1]heptane(compound DC-0040); 1,2-bis(3,4-dihydroxyphenoxy)ethane (compoundDC-0041); 1,3-bis(3,4-dihydroxyphenoxy)propane (compound DC-0042);trans-1,2-bis(3,4-dihydroxyphenoxy)-cyclopentane (compound DC-0043);N-(3,4-dihydroxybenzyl)-3-(3,4-dihydroxyphenoxy)-2-hydroxypropylamine(compound DC-0044); 3,4-dihydroxyphenoxyacetic acid 3,4-dihydroxyanilide(compound DC-0045); 3,4-dihydroxyphenoxyacetic acid3,4-dihydroxybenzylamide (compound DC-0046); 3,4-dihydroxyphenoxyaceticacid 3,4-dihydroxyphenethylamide (compound DC-0047);3,4-dihydroxybenzoic acid p-(3,4-dihydroxyphenoxy)anilide (compoundDC-0048); 3,4-dihydroxybenzoic acid o-(3,4-dihydroxyphenoxy)anilide(compound DC-0049); 2,6-bis(3,4-dihydroxyphenoxy)pyridine (compoundDC-0050), 3,4-dihydroxybenzoic acid 3,4-dihydroxyanilide (compoundDC-0051); 3,4-dihydroxybenzoic acid 3,4-dihydroxybenzylamide (compoundDC-0052); 3,4-dihydroxybenzoic acid 3,4-dihydroxyphenethylamide(compound DC-0053); 3,4-dihydroxyphenylacetic acid 3,4-dihydroxyanilide(compound DC-0054); 3,4-dihydroxyphenylacetic acid3,4-dihydroxybenzylamide (compound DC-0055); 3,4-dihydroxyphenylaceticacid 3,4-dihydroxyphenethylamide (compound DC-0056);3-(3,4-dihydroxyphenyl)propionic acid 3,4-dihydroxyanilide (compoundDC-0057); 3-(3,4-dihydroxyphenyl)propionic acid 3,4-dihydroxybenzylamide(compound DC-0058); 3-(3,4-dihydroxyphenyl)propionic acid3,4-dihydroxyphenethylamide (compound DC-0059); 3,4-dihydroxycinnamicacid 3,4-dihydroxyanilide (compound DC-0060); 3,4-dihydroxycinnamic acid3,4-dihydroxybenzylamide (compound DC-0061); 3,4-dihydroxycinnamic acid3,4-dihydroxyphenethylamide (compound DC-0062); oxalic acidbis(3,4-dihydroxyanilide) (compound DC-0063); oxalic acidbis(3,4-dihydroxybenzylamide) (compound DC-0064); oxalic acidbis(3,4-dihydroxyphenethylamide) (compound DC-0065); succinic acidbis(3,4-dihydroxyanilide) (compound DC-0066); succinic acidbis(3,4-dihydroxybenzylamide) (compound DC-0067); succinic acidbis(3,4-dihydroxyphenethylamide) (compound DC-0068); maleic acidbis(3,4-dihydroxyanilide) (compound DC-0069); maleic acidbis(3,4-dihydroxybenzylamide) (compound DC-0070); fumaric acidbis(3,4-dihydroxyanilide)(compound DC-0071); fumaric acidbis(3,4-dihydroxybenzylamide) (compound DC-0072);bis(3,4-dihydroxybenzyl)amine (compound DC-0073);N-(3,4-dihydroxybenzyl)-3,4-dihydroxyphenethylamine (compound DC-0074);tris(3,4-dihydroxybenzyl)amine (compound DC-0075);1,3-bis(3,4-dihydroxyphenyl)urea (compound DC-0076);1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxybenzyl)urea (compound DC-0077);1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenethyl)urea (compoundDC-0078); 3-deoxy-3-(3,4-dihydroxybenzyl)aminoepicatechin (compoundDC-0079); 3-deoxy-3-(3,4-dihydroxyphenethyl)aminoepicatechin (compoundDC-0080); 2,3,6,7-tetrahydroxy-9,10-epoxy-9,10-dihydroacridine (compoundDC-0081); 10-aminoanthracene-1,2,7,8-tetraol (compound DC-0082);acridine-1,2,6,7-tetraol (compound DC-0083);phenoxazine-2,3,7,8,10-pentaol (compound DC-0084);dibenzo[c,f][2,7]napthyridine-2,3,10,11-tetraol (compound DC-0085); and6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-2,10,11-triol(compound DC-0086);(3) the methylenedioxy analogs and pharmaceutically acceptable esters ofcompounds of (1) and (2); and(4) the pharmaceutically acceptable salts of the compounds of (1) to(3).

Other compounds of the invention for use in the modulation of tauaggregation and dissolution/disruption/inhibition of tau aggregates arecompounds of the formula:

where:R₁ and R₂, and R₃ and R₄ are hydroxyl groups independently positioned atone of the positions selected from the group consisting of 2,3; 2,4;2,5; 2,6; 3,5; 3,6; 4,5; 4,6 and 5,6, andR is selected from a sulfonamide, heteroaryl, tricycloalkyl and —C(O)NR′where R′ is selected from H or CH₃ or pharmaceutically acceptable estersor salts thereof and compounds;

2,3 dihydroxybenzoic acid 3,4 dihydroxyanilide (DC-51-OH1),

3,4 dihydroxybenzoic acid 2,3 dihydroxyanilide (DC-51-OH2),

2,3 dihydroxybenzoic acid 2,3 dihydroxyanilide (DC-51-OH3),

3,4 dihydroxybenzoic acid 3,4 dihydroxy N-methyl anilide (DC-51-CH3),

3,4 dihydroxybenzenesulfonic acid 3,4 dihydroxyphenylsulfonamide(DC-51-W1),

2,4 bis(3,4 dihydroxyphenyl)imidazole (DC-51-W2),

3,5 bis(3,4 dihydroxyphenyl) 1,2,4 triazole (DC-51-W3),

3,5 bis(3,4 dihydroxyphenyl)pyrazole (DC-51-W4),

1,3 bis(3,4 dihydroxyphenyl)adamantane (DC-51-W5).

In a second aspect, this invention is a method of alleviatingtauopathies in a mammal, especially a human, by administration of atherapeutically effective amount of a compound of the first aspect ofthis invention, for example as a pharmaceutical composition.

In a third aspect, this invention is the use of a compound of the firstaspect of this invention in the manufacture of a medicament forallievating tauopathies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A & 1B are photos of Western blots showing that compound DC-51and its analogs modulate levels of TauRD oligomers and monomers ininsoluble fractions of TREx293-TauRDWT.

FIGS. 2A & 2B are photos of Western blots showing that compound DC-51and its analogs modulate levels of TauRD oligomers and monomers insoluble fractions of TREx293-TauRDWT.

FIGS. 3 a and 3 b (panels A-D) are graphs quantifying the relativelevels of soluble/insoluble TauRD oligomers and monomers in compoundDC-51-treated TREx293-TauRDWT cell lysates by quantitative densitometricanalysis of Western blots shown in FIGS. 1&2.

FIGS. 4 a and 4 b (panels A-D) are graphs quantifying the relativelevels of soluble/insoluble TauRD oligomers and monomers in compoundDC-51-W3-treated TREx293-TauRDWT cell lysates by quantitativedensitometric analysis of Western blots shown in FIGS. 1&2.

FIGS. 5 a and 5 b (panels A-D) are graphs quantifying the relativelevels of soluble/insoluble TauRD oligomers and monomers in compoundDC-51-W4-treated TREx293-TauRDWT cell lysates by quantitativedensitometric analysis of Western blots shown in FIGS. 1&2.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In this application, the following terms shall have the followingmeanings, without regard to whether the terms are used varyinglyelsewhere in the literature or otherwise in the known art.

Alleviating or alleviation is used considering the standard dictionarydefinition which is to make something such as pain or hardship morebearable or less severe. With respect to alleviating tauopathies, theperson of skill in the art would understand that the compounds of thisinvention specifically act to reduce or diminish tau protein aggregateswhich are pathological hallmarks of the tauopathies. Thereforealleviating a tauopathy refers to removing or diminishing the occurrenceof pathological hallmarks of the disease.

Chemical structures for each of the compounds of this invention (withthe note that the acetates are shown as representative of thepharmaceutically acceptable esters as a class) are shown in this or theparent application. The names of the compounds are variously IUPAC names[names derived according to the accepted IUPAC (International Union ofPure and Applied Chemistry) system established by the coalition of theCommission on Nomenclature of Organic Chemistry and the Commission onPhysical Organic Chemistry, as can be found athttp://www.chem.qmul.ac.uk/iupact], names derived from IUPAC names byaddition or substitution (for example, by the use of“3,4-methylenedioxyphenyl” derived from “phenyl” instead of“benzo[1,3]dioxol-5-yl”), and names derived from the names of reactants(for example, by the use of “3,4-dihydroxybenzoic acid3,4-dihydroxyanilide” instead of“N-(3,4-dihydroxyphenyl)-3,4-dihydroxybenzamide”). The chemicals withinthe specification will be typically be referred to by DC-##. Thecompounds of the invention are referred to generally as bis andtris-dihydroxyaryl compounds, or sometimes just as “dihydroxyarylcompounds”. It will be noted that compound #84 has an additional hydroxygroup, but does have two dihydroxyaryl groups; while compound #86 hasonly one dihydroxyaryl group but has an additional phenolic hydroxylmoiety.

“Methylenedioxy analogs” refers to the compounds of this invention inwhich each of the pairs of adjacent hydroxyl moieties of thedihydroxyaryl groups have been replaced by methylenedioxy groups. Themethylenedioxy compounds are illustrated and referred to as compounds#1B to #86B or DC-0001B to DC-0086B. The methylenedioxy groups also areconvenient intermediate protecting groups for the dihydroxy moieties andtherefore these disclosed compounds are believed to also serve aseffective prodrugs. The methylenedioxy analogs #1B to #80B areillustrated in Example 30 of the parent application.

“Pharmaceutically acceptable esters” refers to the compounds of thisinvention where the hydroxyl moieties of the dihydroxyaryl groups of thecompounds are esterified with an acid or acids that result in apharmaceutically acceptable poly(ester). The compounds are shown inExample 31 as acetylated, and these acetylated compounds are illustratedand referred to as compounds #1C to #86C or DC-0001C to DC-0086C; but itshould be understood that the depiction of acetyl esters in Example 31of the parent application, is merely illustrative, and allpharmaceutically acceptable esters are included within this invention.The ester groups are expected to serve as intermediate protecting groupsfor the hydroxyl moieties and therefore the pharmaceutically acceptableesters are expected to serve as effective prodrugs for their underlyingbis- and tris-dihydroxyaryl compounds.

“Mammal” includes both humans and non-human mammals, such as companionanimals (cats, dogs, and the like), laboratory animals (such as mice,rats, guinea pigs, and the like) and farm animals (cattle, horses,sheep, goats, swine, and the like).

“Pharmaceutically acceptable excipient” means an excipient that isconventionally useful in preparing a pharmaceutical composition that isgenerally safe, non-toxic, and desirable, and includes excipients thatare acceptable for veterinary use as well as for human pharmaceuticaluse. Such excipients may be solid, liquid, semisolid, or, in the case ofan aerosol composition, gaseous.

“Pharmaceutically acceptable salt” means a salt that is pharmaceuticallyacceptable and have the desired pharmacological properties. Such saltsinclude salts that may be formed where acidic protons present in thecompounds are capable of reacting with inorganic or organic bases.Suitable inorganic salts include those formed with the alkali metals,e.g. sodium and potassium, magnesium, calcium, and aluminum. Suitableorganic salts include those formed with organic bases such as the aminebases, e.g. ethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like. Such salts also include acid additionsalts formed with inorganic acids (e.g. hydrochloric and hydrobromicacids) and organic acids (e.g. acetic acid, citric acid, maleic acid,and the alkane- and arene-sulfonic acids such as methanesulfonic acidand benzenesulfonic acid). When there are two acidic groups present, apharmaceutically acceptable salt may be a mono-acid-mono-salt or adi-salt; and similarly where there are more than two acidic groupspresent, some or all of such groups can be salified.

A “therapeutically effective amount” in general means the amount that,when administered to a subject or animal for alleviating a disease, andis sufficient to affect the desired degree of treatment for the diseaseor reduce or diminish pathological hallmarks associated with thedisease. For example, reducing tau aggregates associated withAlzheimer's disease or another tauopathy. A “therapeutically effectiveamount” or a “therapeutically effective dosage” preferably modulates orinhibits, causes dissolution, and/or disrupts, abnormal tau aggregation,or contributes towards the treatment of a disease associated with theseconditions, such as Alzheimer's disease (AD), Pick's disease (PiD),progressive supranuclear palsy (PSP), corticobasal degeneration (CBD)and familial frontotemporal dementia/Parkinsonism linked to chromosome17 (FTDP-17), amyotrophic lateral sclerosis/Parkinsonism-dementiacomplex, argyrophilic grain dementia, dementia pugilistic, diffuseneurofibrillary tangles with calcification, progressive subcorticalgliosis and tangle only dementia. Effective amounts of a compound ofthis invention or composition thereof for treatment of a mammaliansubject are about 0.1 to about 1000 mg/Kg of body weight of thesubject/day, such as from about 1 to about 100 mg/Kg/day, especiallyfrom about 10 to about 100 mg/Kg/day. A broad range of disclosedcomposition dosages are believed to be both safe and effective.

“Treating” or “treatment” of a disease in general means modulating tauaggregation or the dissolution, disruption, and/or inhibition ofabnormal tau aggregates associated with a tauopathy.

“A pharmaceutical agent” or “pharmacological agent” or “pharmaceuticalcomposition” refers to a compound or combination of compounds used fortreatment, preferably in a pure or near pure form. In the specification,pharmaceutical or pharmacological agents include the compounds of thisinvention. The compounds are desirably purified to 80% homogeneity, andpreferably to 90% homogeneity. Compounds and compositions purified to99.9% homogeneity are believed to be advantageous. As a test orconfirmation, a suitable homogeneous compound on HPLC would yield whatthose skilled in the art would identify as a single sharp-peak band.

Use of compounds of the invention include

(1) compounds of the formula:

where:R is a C₁-C₁₀ alkylene group, in which, when the number of carbon atomsis at least 2, there are optionally 1 or 2 non-adjacent double bonds; 1to 3 non-adjacent methylene groups are optionally replaced by NR′ (whereR′ is H, alkyl, or acyl), O, or S; and 1 or 2 methylene groups areoptionally replaced by a carbonyl or hydroxymethylene group; and(2) the compounds that are:3,4,3′,4′-tetrahydroxybenzoin (compound DC-001);3,4,3′,4′-tetrahydroxydesoxybenzoin (compound DC-002);3,4,3′,4′-tetrahydroxydiphenylmethane (compound DC-003);1,2-bis(3,4-dihydroxyphenyl)ethane (compound DC-004);1,3-bis(3,4-dihydroxyphenyl)propane (compound DC-005);3,4,3′,4′-tetrahydroxychalcone (compound DC-006);3,5-bis(3,4-dihydroxyphenyl)-1-methyl-2-pyrazoline (compound DC-007);4,6-bis(3,4-dihydroxyphenyl)-3-cyano-2-methylpyridine (compound DC-008);1,4-bis(3,4-dihydroxybenzyl)piperazine (compound DC-009);N,N′-bis(3,4-dihydroxybenzyl)-N,N′-dimethylethylenediamine (compoundDC-0010); 2,5-bis(3,4-dihydroxybenzyl)-2,5-diaza[2.2.1]bicycloheptane(compound DC-0011);N,N-bis(3,4-dihydroxybenzyl)-trans-1,2-diaminocyclohexane (compoundDC-0012); N,N′-bis(3,4-dihydroxybenzyl)-trans-1,4-diaminocyclohexane(compound DC-0013);N,N′-bis(3,4-dihydroxybenzyl)-cis-1,3-bis(aminomethyl)cyclohexane(compound DC-0014); N-(3,4-dihydroxybenzyl)proline3,4-dihydroxybenzylamide (compound DC-0015);2-(3,4-dihydroxybenzyl)isoquinoline-3-carboxylic acid3,4-dihydroxyphenethylamide (compound DC-0016);2,6-bis(3,4-dihydroxybenzyl)-cyclohexanone (compound DC-0017);3,5-bis(3,4-dihydroxybenzyl)-1-methyl-4-piperidinone (compound DC-0018);2,4-bis(3,4-dihydroxybenzyl)-3-tropinone (compound DC-0019);tris-(3,4-dihydroxybenzyl)methane (compound DC-0020);α-(3,4-dihydroxybenzamido)-3,4-dihydroxycinnamic acid3,4-dihydroxybenzyl amide (compound DC-0021);4-(3,4-dihydroxybenzylaminomethylene)-2-(3,4-dihydroxyphenyl)oxazolin-5-one(compound DC-0022); 1,4-bis(3,4-dihydroxybenzoyl)piperazine (compoundDC-0023); N,N′-bis(3,4-dihydroxybenzoyl)-N,N′-dimethylethylenediamine(compound DC-0024);2,5-bis(3,4-dihydroxybenzoyl)-2,5-diaza[2.2.1]bicycloheptane (compoundDC-0025); N,N′-bis(3,4-dihydroxybenzoyl)-trans-1,2-diaminocyclohexane(compound DC-0026);N,N′-bis(3,4-dihydroxybenzoyl)-cis-1,3-bis(aminomethyl)cyclohexane(compound DC-0027); 3,6-bis(3,4-dihydroxybenzyl)-2,5-diketopiperazine(compound DC-0028);3,6-bis(3,4-dihydroxybenzylidene)-1,4-dimethyl-2,5-diketopiperazine(compound DC-0029); N-(3,4-dihydroxyphenylacetyl)proline3,4-dihydroxyanilide (compound DC-0030);2,3-bis(3,4-dihydroxyphenyl)butane (compound DC-0031);1,3-bis(3,4-dihydroxybenzyl)benzene (compound DC-0032);1,4-bis(3,4-dihydroxybenzyl)benzene (compound DC-0033);2,6-bis(3,4-dihydroxybenzyl)pyridine (compound DC-0034);2,5-bis(3,4-dihydroxybenzyl)thiophene (compound DC-0035);2,3-bis(3,4-dihydroxybenzyl)thiophene (compound DC-0036);1,2-bis(3,4-dihydroxyphenyl)cyclohexane (compound DC-0037);1,4-bis(3,4-dihydroxyphenyl)cyclohexane (compound DC-0038);3,7-bis(3,4-dihydroxyphenyl)bicyclo[3.3.0]octane (compound DC-0039);2,3-bis(3,4-dihydroxyphenyl)-1,7,7-trimethylbicyclo[2.2.1]heptane(compound DC-0040); 1,2-bis(3,4-dihydroxyphenoxy)ethane (compoundDC-0041); 1,3-bis(3,4-dihydroxyphenoxy)propane (compound DC-0042);trans-1,2-bis(3,4-dihydroxyphenoxy)-cyclopentane (compound DC-0043);N-(3,4-dihydroxybenzyl)-3-(3,4-dihydroxyphenoxy)-2-hydroxypropylamine(compound DC-0044); 3,4-dihydroxyphenoxyacetic acid 3,4-dihydroxyanilide(compound DC-0045); 3,4-dihydroxyphenoxyacetic acid3,4-dihydroxybenzylamide (compound DC-0046); 3,4-dihydroxyphenoxyaceticacid 3,4-dihydroxyphenethylamide (compound DC-0047);3,4-dihydroxybenzoic acid p-(3,4-dihydroxyphenoxy)anilide (compoundDC-0048); 3,4-dihydroxybenzoic acid o-(3,4-dihydroxyphenoxy)anilide(compound DC-0049); 2,6-bis(3,4-dihydroxyphenoxy)pyridine (compoundDC-0050), 3,4-dihydroxybenzoic acid 3,4-dihydroxyanilide (compoundDC-0051); 3,4-dihydroxybenzoic acid 3,4-dihydroxybenzylamide (compoundDC-0052); 3,4-dihydroxybenzoic acid 3,4-dihydroxyphenethylamide(compound DC-0053); 3,4-dihydroxyphenylacetic acid 3,4-dihydroxyanilide(compound DC-0054); 3,4-dihydroxyphenylacetic acid3,4-dihydroxybenzylamide (compound DC-0055); 3,4-dihydroxyphenylaceticacid 3,4-dihydroxyphenethylamide (compound DC-0056);3-(3,4-dihydroxyphenyl)propionic acid 3,4-dihydroxyanilide (compoundDC-0057); 3-(3,4-dihydroxyphenyl)propionic acid 3,4-dihydroxybenzylamide(compound DC-0058); 3-(3,4-dihydroxyphenyl)propionic acid3,4-dihydroxyphenethylamide (compound DC-0059); 3,4-dihydroxycinnamicacid 3,4-dihydroxyanilide (compound DC-0060); 3,4-dihydroxycinnamic acid3,4-dihydroxybenzylamide (compound DC-0061); 3,4-dihydroxycinnamic acid3,4-dihydroxyphenethylamide (compound DC-0062); oxalic acidbis(3,4-dihydroxyanilide) (compound DC-0063); oxalic acidbis(3,4-dihydroxybenzylamide) (compound DC-0064); oxalic acidbis(3,4-dihydroxyphenethylamide) (compound DC-0065); succinic acidbis(3,4-dihydroxyanilide) (compound DC-0066); succinic acidbis(3,4-dihydroxybenzylamide) (compound DC-0067); succinic acidbis(3,4-dihydroxyphenethylamide) (compound DC-0068); maleic acidbis(3,4-dihydroxyanilide) (compound DC-0069); maleic acidbis(3,4-dihydroxybenzylamide) (compound DC-0070); fumaric acidbis(3,4-dihydroxyanilide)(compound DC-0071); fumaric acidbis(3,4-dihydroxybenzylamide) (compound DC-0072);bis(3,4-dihydroxybenzyl)amine (compound DC-0073);N-(3,4-dihydroxybenzyl)-3,4-dihydroxyphenethylamine (compound DC-0074);tris(3,4-dihydroxybenzyl)amine (compound DC-0075);1,3-bis(3,4-dihydroxyphenyl)urea (compound DC-0076);1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxybenzyl)urea (compound DC-0077);1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenethyl)urea (compoundDC-0078); 3-deoxy-3-(3,4-dihydroxybenzyl)aminoepicatechin (compoundDC-0079); 3-deoxy-3-(3,4-dihydroxyphenethyl)aminoepicatechin (compoundDC-0080); 2,3,6,7-tetrahydroxy-9,10-epoxy-9,10-dihydroacridine (compoundDC-0081); 10-aminoanthracene-1,2,7,8-tetraol (compound DC-0082);acridine-1,2,6,7-tetraol (compound DC-0083);phenoxazine-2,3,7,8,10-pentaol (compound DC-0084);dibenzo[c,f][2,7]napthyridine-2,3,10,11-tetraol (compound DC-0085); and6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-2,10,11-triol(compound DC-0086);(3) the methylenedioxy analogs and pharmaceutically acceptable esters ofcompounds of (1) and (2); and(4) the pharmaceutically acceptable salts of the compounds of (1) to(3).

Other compounds of the invention for use are:

compounds of the formula:

where:R₁ and R₂, and R₃ and R₄ are hydroxyl groups independently positioned atone of the positions selected from the group consisting of 2,3; 2,4;2,5; 2,6; 3,5; 3,6; 4,5; 4,6 and 5,6, andR is selected from a sulfonamide, heteroaryl, tricycloalkyl and —C(O)NR′where R′ is selected from H or CH₃ or pharmaceutically acceptable estersor salts thereof.

The compounds of this invention for use are also:

2,3 dihydroxybenzoic acid 3,4 dihydroxyanilide (DC-51-OH1),

3,4 dihydroxybenzoic acid 2,3 dihydroxyanilide (DC-51-OH2),

2,3 dihydroxybenzoic acid 2,3 dihydroxyanilide (DC-51-OH3),

3,4 dihydroxybenzoic acid 3,4 dihydroxy N-methyl anilide (DC-51-CH3),

3,4 dihydroxybenzenesulfonic acid 3,4 dihydroxyphenylsulfonamide(DC-51-W1),

2,4 bis(3,4 dihydroxyphenyl)imidazole (DC-51-W2),

3,5 bis(3,4 dihydroxyphenyl) 1,2,4 triazole (DC-51-W3),

3,5 bis(3,4 dihydroxyphenyl)pyrazole (DC-51-W4),

1,3 bis(3,4 dihydroxyphenyl)adamantane (DC-51-W5),

Synthesis of the Compounds of the Invention

The compounds of this invention may be prepared by methods generallyknown to the person of ordinary skill in the art, having regard to thatknowledge and the disclosure of this application and its priorityapplications, the contents of which are incorporated by reference.

The starting materials and reagents used in preparing these compoundsare either available from commercial suppliers such as the AldrichChemical Company (Milwaukee, Wis.), Bachem (Torrance, Calif.), Sigma(St. Louis, Mo.), or Lancaster Synthesis Inc. (Windham, N.H.) or areprepared by methods well known to a person of ordinary skill in the art,following procedures described in such references as Fieser and Fieser'sReagents for Organic Synthesis, vols. 1-17, John Wiley and Sons, NewYork, N.Y., 1991; Rodd's Chemistry of Carbon Compounds, vols. 1-5 andsupps., Elsevier Science Publishers, 1989; Organic Reactions, vols.1-40, John Wiley and Sons, New York, N.Y., 1991; March J.: AdvancedOrganic Chemistry, 4th ed., John Wiley and Sons, New York, N.Y.; andLarock: Comprehensive Organic Transformations, VCH Publishers, New York,1989.

Other starting materials or early intermediates may be prepared byelaboration of the materials listed above, for example, by methods wellknown to a person of ordinary skill in the art.

The starting materials, intermediates, and compounds of this inventionmay be isolated and purified using conventional techniques, includingprecipitation, filtration, distillation, crystallization,chromatography, and the like. The compounds may be characterized usingconventional methods, including physical constants and spectroscopicmethods.

Pharmacology and Utility

The use of the compounds of this invention, either as the dihydroxyarylcompounds per se, or as the methylenedioxy analogs or pharmaceuticallyacceptable esters (once de-protected either in the body or in vitro), orthe compounds set out herein to modulate tau aggregation or to cause thedissolution, disruption and/or inhibition of tau aggregates and foralleviating tauopathies. Their activity can be measured in vitro bymethods such as those discussed in Example 1, while their activity invivo against tauopathies can be measured in animal models, such as thosetransgenic mouse models that mimic AD and other tauopathies, and inhumans (Dickey, C et al., 2009 Am J. Pathol. 174(1):228-38; Boimel, M etal., 2009 J Neuropathol Exp Neurol 68(3):314-25; and Lopes, J P et al.,2009 J Alzh Dis 16(3):541-9.)

“Tauopathies” suitable for alleviation with the compounds of thisinvention are diseases associated with abnormal tau aggregation areAlzheimer's disease (AD), Pick's disease (PiD), progressive supranuclearpalsy (PSP), corticobasal degeneration (CBD) and familial frontotemporaldementia/Parkinsonism linked to chromosome 17 (FTDP-17), amyotrophiclateral sclerosis/Parkinsonism-dementia complex, argyrophilic graindementia, dementia pugilistic, diffuse neurofibrillary tangles withcalcification, progressive subcortical gliosis and tangle only dementia.

Pharmaceutical Compositions and Administration

In general, compounds of the invention will be administered intherapeutically effective amounts by any of the usual modes known in theart, either singly or in combination with at least one other compound ofthis invention and/or at least one other conventional therapeutic agentfor the disease being treated. A therapeutically effective amount mayvary widely depending on the disease, its severity, the age and relativehealth of the animal being treated, the potency of the compound(s), andother factors. As tau protein modulators or aggregation inhibitors,therapeutically effective amounts of compounds of this invention mayrange from 0.1-1000 mg/Kg body weight/day, such as from 1-100 mg/Kg/day;for example, 10-100 mg/Kg/day. A person of ordinary skill in the artwill be conventionally able, and without undue experimentation, havingregard to that skill and to this disclosure, to determine atherapeutically effective amount of a compound for the alleviation oftau aggregation or tauopathy.

Preferred compositions will contain a compound of this invention that isat least substantially pure. In general “pure” means better than 95%pure, and “substantially pure” means a compound synthesized such thatthe compound, as made as available for consideration into a therapeuticdosage, has only those impurities that can not readily nor reasonably beremoved by conventional purification processes.

In general, the compounds of this invention will be administered aspharmaceutical compositions by one of the following routes: oral,topical, systemic (e.g. transdermal, intranasal, or by suppository), orparenteral (e.g. intramuscular, subcutaneous, or intravenous injection).Compositions may take the form of tablets, pills, capsules, semisolids,powders, sustained release formulations, solutions, suspensions,elixirs, aerosols, or any other appropriate compositions; and compriseat least one compound of this invention in combination with at least onepharmaceutically acceptable excipient. Suitable excipients are wellknown to persons of ordinary skill in the art, and they, and the methodsof formulating the compositions, may be found in such standardreferences as Remington: The Science and Practice of Pharmacy, A.Gennaro, ed., 20th edition, Lippincott, Williams & Wilkins,Philadelphia, Pa. Suitable liquid carriers, especially for injectablesolutions, include water, aqueous saline solution, aqueous dextrosesolution, and glycols.

In particular, the compound(s)—optimally only one such compound isadministered in any particular dosage form—can be administered, orally,for example, as tablets, troches, lozenges, aqueous or oily suspension,dispersible powders or granules, emulsions, hard or soft capsules, orsyrups or elixirs. Compositions intended for oral use may be preparedaccording to any method known in the art for the manufacture ofpharmaceutical compositions and such compositions may contain one ormore agents selected from the group consisting of sweetening agents,flavoring agents, coloring agents and preserving agents in order toprovide pharmaceutically elegant and palatable preparations.

Tablets contain the compound in admixture with non-toxicpharmaceutically acceptable excipients that are suitable for themanufacture of tablets. These excipients may be for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, maize starch or alginic acid; binding agents, for example,maize starch, gelatin or acacia, and lubricating agents, for example,magnesium stearate or stearic acid or tale. The tablets may be uncoatedor they may be coated by known techniques to delay disintegration andabsorption in the gastrointestinal tract and thereby provide a sustainedaction over a longer period. For example, a time delay material such asglycerol monostearate or glycerol distearate may be employed.Formulations for oral use may also be presented as hard gelatin capsuleswherein the compound is mixed with an inert solid diluent, for example,calcium carbonate, calcium phosphate or kaolin, or as soft gelatincapsules wherein the active ingredient is mixed with water or an oilmedium, for example, peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the compound in admixture with excipientssuitable for the manufacture of aqueous suspensions. Such excipients aresuspending agents, for example, sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethyl cellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing orwetting agents may be naturally occurring phosphatides, for examplelecithin, or condensation products of an alkylene oxide with fattyacids, for example polyoxyethylene stearate, or condensation products ofethylene oxide with long chain aliphatic alcohols, for example,heptadecaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids such as hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters from fatty acids and a hexitolanhydrides, for example, polyethylene sorbitan monooleate. The aqueoussuspensions may also contain one or more preservatives, for example,ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents, or one or more sweetening agents, such as sucroseor saccharin.

Oily suspensions may be formulated by suspending the compound in avegetable oil, for example arachis oil, olive oil, sesame oil, orcoconut oil or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents, such as those set forthbelow, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of anantioxidant such as ascorbic acid. Dispersible powders and granulessuitable for preparation of an aqueous suspension by the addition ofwater provide the active ingredient in admixture with a dispersing orwetting agent, a suspending agent and one or more preservatives.Suitable dispersing or wetting agents and suspending agents areexemplified by those already described above. Additional excipients, forexample sweetening, flavoring and agents, may also be present.

The compounds of the invention may also be in the form of oil-in-wateremulsions. The oily phase may be a vegetable oil, for example olive oilor arachis oils, or a mineral oil, for example liquid paraffin ormixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally occurring phosphatides, for example soy bean, lecithin, andoccurring phosphatides, for example soy bean, lecithin, and esters orpartial esters derived from fatty acids and hexitol anhydrides, forexample sorbitan monooleate, and condensation products of the saidpartial esters with ethylene oxide, for example polyoxyethylene sorbitanmonooleate. The emulsion may also contain sweetening and flavoringagents. Syrups and elixirs may be formulated with sweetening agents, forexample, glycerol, sorbitol or sucrose. Such formulations may alsocontain a demulcent, a preservative and flavoring and coloring agents.

The compounds of the invention can also be administered by injection orinfusion, either subcutaneously or intravenously, or intramuscularly, orintrasternally, or intranasally, or by infusion techniques in the formof sterile injectable or oleaginous suspension. The compound may be inthe form of a sterile injectable aqueous or oleaginous suspensions.These suspensions may be formulated according to the known art usingsuitable dispersing of wetting agents and suspending agents that havebeen described above. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilsmay be conventionally employed including synthetic mono- ordiglycerides. In addition fatty acids such as oleic acid find use in thepreparation of injectables. Dosage regimens can be adjusted to providethe optimum therapeutic response. For example, several divided dosagesmay be administered daily or the dosage may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

It is especially advantageous to formulate the compounds in dosage unitform for ease of administration and uniformity of dosage. Dosage unitform as used herein refers to physically discrete units suited asunitary dosages for the subjects to be treated; each containing atherapeutically effective quantity of the compound and at least onepharmaceutical excipient. A drug product will comprise a dosage unitform within a container that is labeled or accompanied by a labelindicating the intended method of treatment, such as the treatment of atauopathy or disease associated with abnormal tau aggregation.

Sustained Release Formulations

The invention also includes the use of sustained release formulations todeliver the compounds of the present invention to the desired target(i.e. brain or systemic organs) at high circulating levels (between 10⁻⁹and 10⁻⁴ M) are also disclosed. In a preferred embodiment for thetreatment of Alzheimer's or other tauopathies, the circulating levels ofthe compounds is maintained up to 10⁻⁷ M. The levels are eithercirculating in the patient systemically, or in a preferred embodiment,present in brain tissue, and in a most preferred embodiment, localizedto the areas of abnormal tau aggregation.

It is understood that the compound levels are maintained over a certainperiod of time as is desired and can be easily determined by one skilledin the art using this disclosure and compounds of the invention. In apreferred embodiment, the invention includes a unique feature ofadministration comprising a sustained release formulation so that aconstant level of therapeutic compound is maintained between 10⁻⁸ and10⁻⁶ M between 48 to 96 hours in the sera.

Such sustained and/or timed release formulations may be made bysustained release means of delivery devices that are well known to thoseof ordinary skill in the art, such as those described in U.S. Pat. Nos.3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 4,710,384;5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476;5,354,556 and 5,733,566, the disclosures of which are each incorporatedherein by reference. These pharmaceutical compositions can be used toprovide slow or sustained release of one or more of the active compoundsusing, for example, hydroxypropylmethyl cellulose, other polymermatrices, gels, permeable membranes, osmotic systems, multilayercoatings, microparticles, liposomes, microspheres, or the like. Suitablesustained release formulations known to those skilled in the art,including those described herein, may be readily selected for use withthe pharmaceutical compositions of the invention. Thus, single unitdosage forms suitable for oral administration, such as, but not limitedto, tablets, capsules, gelcaps, caplets, powders and the like, that areadapted for sustained release are encompassed by the present invention.

In a preferred embodiment, the sustained release formulation containsactive compound such as, but not limited to, microcrystalline cellulose,maltodextrin, ethylcellulose, and magnesium stearate. As describedabove, all known methods for encapsulation which are compatible withproperties of the disclosed compounds are encompassed by this invention.The sustained release formulation is encapsulated by coating particlesor granules of the pharmaceutical composition of the invention withvarying thickness of slowly soluble polymers or by microencapsulation.In a preferred embodiment, the sustained release formulation isencapsulated with a coating material of varying thickness (e.g. about 1micron to 200 microns) that allow the dissolution of the pharmaceuticalcomposition about 48 hours to about 72 hours after administration to amammal. In another embodiment, the coating material is a food-approvedadditive.

In another embodiment, the sustained release formulation is a matrixdissolution device that is prepared by compressing the drug with aslowly soluble polymer carrier into a tablet. In one preferredembodiment, the coated particles have a size range between about 0.1 toabout 300 microns, as disclosed in U.S. Pat. Nos. 4,710,384 and5,354,556, which are incorporated herein by reference in theirentireties. Each of the particles is in the form of a micromatrix, withthe active ingredient uniformly distributed throughout the polymer.

Sustained release formulations such as those described in U.S. Pat. No.4,710,384, which is incorporated herein by reference in its entirety,having a relatively high percentage of plasticizer in the coating inorder to permit sufficient flexibility to prevent substantial breakageduring compression are disclosed. The specific amount of plasticizervaries depending on the nature of the coating and the particularplasticizer used. The amount may be readily determined empirically bytesting the release characteristics of the tablets formed. If themedicament is released too quickly, then more plasticizer is used.Release characteristics are also a function of the thickness of thecoating. When substantial amounts of plasticizer are used, the sustainedrelease capacity of the coating diminishes. Thus, the thickness of thecoating may be increased slightly to make up for an increase in theamount of plasticizer. Generally, the plasticizer in such an embodimentwill be present in an amount of about 15 to 30% of the sustained releasematerial in the coating, preferably 20 to 25%, and the amount of coatingwill be from 10 to 25% of the weight of the active material, preferably15 to 20%. Any conventional pharmaceutically acceptable plasticizer maybe incorporated into the coating.

The compounds of the invention can be formulated as a sustained and/ortimed release formulation. All sustained release pharmaceutical productshave a common goal of improving drug therapy over that achieved by theirnon-sustained counterparts. Ideally, the use of an optimally designedsustained release preparation in medical treatment is characterized by aminimum of drug substance being employed to cure or control thecondition. Advantages of sustained release formulations may include: 1)extended activity of the composition, 2) reduced dosage frequency, and3) increased patient compliance. In addition, sustained releaseformulations can be used to affect the time of onset of action or othercharacteristics, such as blood levels of the composition, and thus canaffect the occurrence of side effects.

The sustained release formulations of the invention are designed toinitially release an amount of the therapeutic composition that promptlyproduces the desired therapeutic effect, and gradually and continuallyrelease of other amounts of compositions to maintain this level oftherapeutic effect over an extended period of time. In order to maintainthis constant level in the body, the therapeutic composition must bereleased from the dosage form at a rate that will replace thecomposition being metabolized and excreted from the body.

The sustained release of an active ingredient may be stimulated byvarious inducers, for example pH, temperature, enzymes, water, or otherphysiological conditions or compounds. The term “sustained releasecomponent” in the context of the present invention is defined herein asa compound or compounds, including, but not limited to, polymers,polymer matrices, gels, permeable membranes, liposomes, microspheres, orthe like, or a combination thereof, that facilitates the sustainedrelease of the active ingredient.

If the complex is water-soluble, it may be formulated in an appropriatebuffer, for example, phosphate buffered saline, or other physiologicallycompatible solutions. Alternatively, if the resulting complex has poorsolubility in aqueous solvents, then it may be formulated with anon-ionic surfactant such as Tween, or polyethylene glycol. Thus, thecompounds and their physiologically solvents may be formulated foradministration by inhalation or insufflation (either through the mouthor the nose) or oral, buccal, parenteral, or rectal administration, asexamples.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound. In a preferred embodiment,the compounds of the present invention are formulated as controlledrelease powders of discrete microparticles that can be readilyformulated in liquid form. The sustained release powder comprisesparticles containing an active ingredient and optionally, an excipientwith at least one non-toxic polymer.

The powder can be dispersed or suspended in a liquid vehicle and willmaintain its sustained release characteristics for a useful period oftime. These dispersions or suspensions have both chemical stability andstability in terms of dissolution rate. The powder may contain anexcipient comprising a polymer, which may be soluble, insoluble,permeable, impermeable, or biodegradable. The polymers may be polymersor copolymers. The polymer may be a natural or synthetic polymer.Natural polymers include polypeptides (e.g., zein), polysaccharides(e.g., cellulose), and alginic acid. Representative synthetic polymersinclude those described, but not limited to, those described in column3, lines 33-45 of U.S. Pat. No. 5,354,556, which is incorporated byreference in its entirety. Particularly suitable polymers include thosedescribed, but not limited to those described in column 3, line46-column 4, line 8 of U.S. Pat. No. 5,354,556 which is incorporated byreference in its entirety.

The sustained release compounds of the invention may be formulated forparenteral administration, e.g., by intramuscular injections or implantsfor subcutaneous tissues and various body cavities and transdermaldevices. In one embodiment, intramuscular injections are formulated asaqueous or oil suspensions. In an aqueous suspension, the sustainedrelease effect is due to, in part, a reduction in solubility of theactive compound upon complexation or a decrease in dissolution rate. Asimilar approach is taken with oil suspensions and solutions, whereinthe release rate of an active compound is determined by partitioning ofthe active compound out of the oil into the surrounding aqueous medium.Only active compounds which are oil soluble and have the desiredpartition characteristics are suitable. Oils that may be used forintramuscular injection include, but are not limited to, sesame, olive,arachis, maize, almond, soybean, cottonseed and castor oil.

A highly developed form of drug delivery that imparts sustained releaseover periods of time ranging from days to years is to implant adrug-bearing polymeric device subcutaneously or in various bodycavities. The polymer material used in an implant, which must bebiocompatible and nontoxic, include but are not limited to hydrogels,silicones, polyethylenes, ethylene-vinyl acetate copolymers, orbiodegradable polymers.

The following non-limiting Examples are given by way of illustrationonly and are not considered a limitation of this invention, manyapparent variations of which are possible without departing from thespirit or scope thereof.

EXAMPLES Example 1 Compounds of this Invention are Potent Disrupters ofTau Aggregates

The compounds set out above were found to be potent in thedissolution/disruption/inhibition of Tau tangles or Tau aggregates. In aset of studies, the efficacy of the compounds to cause adissolution/disassembly/disruption of pre-formed Tau aggregates wasanalyzed.

Part A—Thioflavin T Fluorometry Data

Thioflavin T fluorometry was used in this study to determine the effectsof the compounds compared to a negative control peptide. Thioflavin Tbinds specifically to aggregated Tau or Tau tangles, and this bindingproduces a fluorescence enhancement at 485 nm that is directlyproportional to the amount of aggregated Tau. The higher thefluorescence, the greater the amount of aggregated Tau.

In this study, Tau-441 was pre-fibrillized or aggregated by combiningwith Heparin (SIGMA) at 1:1 wt/wt, then incubation at 37° C. and shakingat 1400 rpm for 8 days. Following the pre-fibrillization, 30 μg ofaggregated Tau-441 (rPeptide) was then incubated at 37° C. for 3 dayseither alone, or in the presence of one of the compounds or negativecontrol (at Tau:test compound weight ratios of 1:1, 1:0.1, 1:0.01 or1:0.001). Following 3-days of co-incubation, 50 μl of each incubationmixture was transferred into a 96-well microliter plate containing 150μl of distilled water and 50 p. 1 of a Thioflavin T solution (i.e. 500mM Thioflavin T in 250 mM phosphate buffer, pH 6.8). The fluorescencewas read at 485 nm (444 nm excitation wavelength) using an ELISA platefluorometer after subtraction with buffer alone or compound alone, asblank.

The results are presented in the table below. For example, as expected,the negative control caused no significantdissolution/disruption/inhibition of preformed Tau aggregates at all ofthe concentrations tested, in contrast, the compounds all caused adose-dependent dissolution/disruption/inhibition of preformed Tauaggregates. All the compounds tested, with the exception of DC-0023 andDC-0063, were highly efficacious in their ability to disrupt preformedTau aggregates. For example, DC-5′-W2 caused a significant (p<0.01)92.4±1.23% dissolution/disruption/inhibition when used at a Tau:testcompound wt/wt ratio of 1:0.1, and a 74.0±4.01%dissolution/disruption/inhibition when used at a Tau: compound wt/wtratio of 1:0.01. Under the same conditions (i.e. Tau:test compound wt/wtratio of 1:0.1), compound DC-004 caused a 92.8±2.59%dissolution/disruption/inhibition, and a 68.0±1.71%dissolution/disruption/inhibition when used at a Tau:compound wt/wtratio of 1:0.01. This study indicated that the compounds of thisinvention can result in the potent dissolution, disruption and/orinhibition of Tau aggregates, and typically exert their effects in adose-dependent manner.

TABLE 1 Thioflavin T fluorometry data -dissolution/disruption/inhibition of Tau aggregates %dissolution/disruption/inhibition Tau (result ± S.D.) at Tau:testcompound wt/wt ratio Test Compound # 1:1 1:0.1 1:0.01 1:0.001 Negative15.4 ± 3.97 11.5 ± 2.85 11.2 ± 3.47 11.1 ± 1.98 control DC-003 91.2 ±2.59 89.2 ± 1.96 54.1 ± 2.62 17.0 ± 2.49 DC-004 93.5 ± 4.42 92.8 ± 2.5968.0 ± 1.71 19.3 ± 0.00 DC-0021 94.8 ± 2.59 75.2 ± 2.04 31.7 ± 5.45  9.8± 2.95 DC-0023 71.3 ± 2.04 33.4 ± 1.49 15.2 ± 2.49 11.4 ± 1.14 DC-005196.1 ± 0.56 87.9 ± 5.03 51.5 ± 5.51 11.8 ± 4.67 DC-0051-C 92.2 ± 2.0481.7 ± 4.07 38.6 ± 6.59  5.2 ± 6.04 DC-0063 77.8 ± 1.13 34.7 ± 1.69 16.5± 2.49 11.8 ± 8.06 DC-0076 94.1 ± 2.99 91.2 ± 0.56 54.1 ± 2.62 17.4 ±4.88 DC-51-OH1 100.0 ± 1.62  90.0 ± 2.14 58.0 ± 2.05 13.2 ± 5.38DC-51-OH2 100.0 ± 3.38  88.1 ± 2.85 43.7 ± 3.38 10.6 ± 5.36 DC-51-OH3100.0 ± 2.94  86.0 ± 5.69 61.5 ± 2.05 12.4 ± 5.38 DC-51-CH3 100.0 ±0.89  90.0 ± 2.43 59.4 ± 2.61 13.0 ± 5.06 DC-51-W1 100.0 ± 1.55  87.3 ±1.69 50.2 ± 4.96 16.5 ± 2.48 DC-51-W2 97.7 ± 1.95 92.4 ± 1.23 74.0 ±4.01 25.7 ± 4.90 DC-51-W3 100.0 ± 1.62  90.3 ± 2.04 37.2 ± 4.96  6.0 ±5.12 DC-51-W4 100.0 ± 4.32  88.9 ± 6.74 57.7 ± 24.96 11.9 ± 3.28DC-51-W5 96.6 ± 2.80 91.1 ± 0.81 35.2 ± 2.59  7.6 ± 3.38

Example 2 Cloning of Tau Repeat Domains (TauRD) into aTetracycline-Inducible Mammalian Expression Vector

Total RNA was isolated from human adult non-demented frontal tissuesobtained at autopsy from the University of Washington ADRC Brain Bankand immediately frozen at 80° C. Single stranded cDNA was synthesizedusing M-MLV Reverse Transcriptase (Invitrogen; Carlsbad, Calif., USA)and random priming with hexameric primers (Invitrogen). All otherprimers used were also synthesized by Invitrogen. Tetracycline-induciblemammalian expression constructs, pcDNA4/TO-TauRD, were generated byinsertion of cDNA fragments encoding the human tau repeat domain (TauRD;amino acid residues 244-372 in REFSEQ mRNA ENST00000351559) intopcDNA™4/TO vector (Invitrogen). The vector allows tetracycline-regulatedexpression of the gene of interest in mammalian host cells when apcDNA™6/TR construct (Invitrogen) is also co-expressed.

The wild type TauRD (TauRDWT) cDNA insert was amplified from human brainsingle stranded cDNA by PCR with a forward primer (Tau6+730F),overlapped with the cDNA sequences of residues 244-249, and a reverseprimer (Tau1116+3R) overlapped with residues 364-372 of human tau(REFSEQ mRNA ENST00000351559). The PCR products were first cloned into apDrive-UA cloning vector (QIAGEN; Valencia, Calif., USA) as instructedby the manufacturer to generate pDrive-TauRDWT. Mutant constructs thatcontain mutations of ΔK280, P301S, and P301L found in frontotemporaldementia with Parkinsonism-17 were made on the pDrive-TauRDWT backboneusing a QuikChange® Site-Directed Mutagenesis Kit (Stratagene, La Jolla,Calif.), as instructed by the manufacturer. The mutations have beenshown to increase Tau aggregations in vitro or in vivo (Brunden et al.,2009). After confirmed by DNA sequencing, the cDNA inserts inpDrive-TauRDWT, pDrive-TauRDΔK280, pDrive-TauRDP301S andpDrive-TauRDP301L were then released by EcoRI digestion of the plasmids,gel-purified with a gel extraction kit (QIAGEN) as instructed, andsubcloned into a pcDNA™4/TO vector at EcoRI sites to generatepcDNA4/TO-TauRDWT, pcDNA4/TO-TauRDΔK280, pcDNA4/TO-TauRDP301S, andpcDNA4/TO-TauRDP301L expression constructs. The pcDNA4/TO-TauRDconstructs are driven by a hybrid promoter consisting of the humancytomegalovirus immediate-early promoter and tetracycline operator 2sites for high-level tetracycline-regulated expression in mammaliancells. In the absence of tetracycline, expression of the inserted TauRDis repressed by the binding of Tet repressor homodimers (expressed frompcDNA™6/TR) to the tetracycline operator 2 sites. Addition oftetracycline to the cells de-represses the hybrid promoter inpcDNA™4/TO, and allows expression of TauRD. Because expression of TauRDcan form aggregates in the cells, long-term expression of aggregatedTauRD may be toxic to cells (Khlistunova et al., 2006 JBC281(2):1205-14). Use of the tetracycline-regulated constructs allows usto generate stable cell lines in which expression of TauRD is under thecontrol by addition of tetracycline into cell culture media.

Example 3 Generation of Stable Transfected Inducible TauRD Cell Lines

Tetracycline-inducible cell lines stably transfected withpcDNA4/TO-TauRDWT, pcDNA4/TO-TauRDΔK280, pcDNA4/TO-TauRDP301S, andpcDNA4/TO-TauRDP301L were generated to assess TauRD aggregation in cellculture. TREx™-293 cells (Invitrogen; R710-07), modified human embryonickidney 293 cells (ATCC; CRL-1573) by stable transfection of pcDNA™6/TR,were employed to generate the TauRD stable cell lines. Both parentalcells (TREx™-293 cells) and their derivatives (TREx293-TauRD) (seebelow) were maintained in culture media supplemented with 5 μg/mlblasticidin for selection of pcDNA6/TR-containing cells. Cells wereroutinely cultured in a regular growth media (RGM) that containedDulbecco's Modified Eagle Medium (DMEM) (Invitrogen) supplemented with10% fetal bovine serum, penicillin (60 units/mL), streptomycin (60μg/mL) and blasticidin (5) at 37° C. in a cell culture incubatorsupplemented with 5% CO₂.

To generate stable cell lines, TREx™-293 cells were grown to 70-80%confluence in 12-well plates, and transfected with 1.2 μg ofpcDNA4/TO-TauRDWT, pcDNA4/TO-TauRDΔK280, pcDNA4/TO-TauRDP301S, orpcDNA4/TO-TauRDP301L. Transfection was mediated by polyethylenimines(Polysciences, Inc.) as described by Hu et al. (2005 JBC280(13):12548-58). Twenty-four hours after transfection, cells wereplated at low density (400-2000 cells/100 mm dish), and grown in RGMcontaining 0.4 mg/ml of Zeocin (Invitrogen) to selectpcDNA4/TO-TauRD-containing stable colonies. After two weeks, stablecolonies were picked, and sub-cultured. Stable expression of TauRD wasconfirmed by Western analysis of cell lysates with a rabbit anti-humanTau polyclonal antibody (DakoCytomation, Denmark), a mouse anti-Taumonoclonal antibody (DC4R; rPeptide, Bogart, Ga.), and/or a rabbitanti-pTau (Ser 262) (Santa Cruz Biotechnology, Inc., Santa Cruz,Calif.). The stable cell lines were maintained with Zeocin-containingRGM. TREx™-293 cells stably transfected with various pcDNA4/TO-TauRDconstructs are referred as TREx293-TauRDWT, TREx293-TauRDΔK280,TREx293-TauRDP301S, and TREx293-TauRDP301L cells.

Example 4 Western Analysis of Soluble/Insoluble TauRD Monomers andOligomers in Cell Lysates

Soluble/insoluble TauRD monomers and oligomers in lysates were preparedand analyzed as follows. TREx293-TauRDWT (clone B1) cells were grown inRGM supplemented with 0.4 mg/ml of Zeocin, 1 μg/ml of tetracycline(Tet+), and with or without addition of compounds for 2 days. Cells weregently washed once with PBS, and were lysed in a cold lysis buffer 50 mMTris-HCl, pH 7.5, 100 mM NaCl, and 0.5% Triton X-100, supplemented withComplete™ protease inhibitor cocktail tablets (Roche) at one tablet/25ml lysis buffer on ice for 20 min. Cell lysates were collected with cellscrapers into centrifuge tubes, and centrifuged at 15,000×g at 4° C. for15 min. The supernatants were collected as soluble fractions. Pelletswere then dissolved in non-reduced 1× Laemmli buffer [2% sodium dodecylsulfate (SDS), 10% glycerol, 0.002% bromphenol blue, 62.5 mM Tris HCl,pH 6.8] by shaking at 24° C. for 50 min, boiling for 5 min, andcentrifuging at 15,000×g at 4° C. for 15 min. The supernatants werecollected as insoluble fractions and were directly loaded on Westerngels; the soluble fractions were diluted at 3:1 in 4× non-reducedLaemmli buffer before loading on Western gels.

Soluble/insoluble fractions of cell lysates were separated in 4-12%Bis/Tris Criterion XT gels (Bio-Rad) at 180 volts, with buffer systemsrecommended by the manufacturer. After electrophoresis, protein bandswere transferred onto Immobilon-PSQ membranes in a tris/glycine transferbuffer (Bio-Rad) using Bio-Rad Criterion Blotters. The transfer wasconducted at 0.25 A (constant) for 60 min at 4° C. All transferredmembranes were blocked with 5% milk in PBS+0.05% Tween-20 for 30-60 minat room temperature, and incubated with a rabbit anti-human Taupolyclonal antibody (DakoCytomation, Denmark) at 1:10000 for overnightat 4° C., and then with HRP-conjugated secondary antibody (Vector) at1:4000 at room temperature for 2 hrs. Protein bands were visualized withan ECL system (GE Healthcare) by exposing to autoradiography films. Forre-probing membranes with different antibodies, membranes were strippedwith a Restore™ Western blot stripping buffer (Thermo Scientific;Rockford, Ill.), and re-probed with mouse anti-α-tubulin (1:30,000)(Sigma; Saint Louis, Mo.), and/or anti-β-actin (1:200,000) (ChemiconInternational) monoclonal antibodies. Quantitation of relativeintensities of protein bands on autoradiographic films was performed byimage quantification with the ScionImage software(http://www.scioncorp.com) as instructed in the product manual.

Example 5 Treatment of Cultured Cells with Compound DC-51 and itsAnalogs

Stock solutions (100 mM) of the compounds were prepared in dimethylsulfoxide (DMSO), aliquoted and stored at −80° C. before use. On the daybefore treatment, TREx293-TauRDWT (clone B1) cells were plated in12-well culture plates with RGM, supplemented with 0.4 mg/ml of Zeocin.The plating density was optimized to reach 25-30% of confluence on nextday. On the next day, cell culture media was replaced with 1 ml per wellof RGM (eliminating blasticidin) supplemented with 1 μg/ml oftetracycline (Tet+), and freshly-diluted test compounds at the finalconcentrations between 0.25-100 M. Cells were then incubated at 37° C.in a cell culture incubator for total 48 hrs, with freshtetracycline/compound-containing media changed every 24 hrs. Afterincubation, cell lysates were collected for Western analysis ofaggregated TauRD as described.

Example 6 Effects of Compound DC-51 and its Analogs on Aggregation andSolubility of TauRD in Cultured Cells as Assessed by Western Analysis

FIG. 1 shows that compound DC-51 and its analogs modulate levels ofTauRD oligomers and monomers in insoluble fractions of TREx293-TauRDWT(clone B1) cell cultures as assessed by Western analysis.TREx293-TauRDWT cells were treated with DMSO vehicle control (Lanes 2,15 and 28), compound DC-51 (0.5 μM in lanes 9&12; 1 μM in lanes 10&13;10 μM in lanes 11&14) and its analogs DC-51-W3 (10 μM in lanes 16&19; 40μM in lanes 17&20; 80 μM in lanes 18&21), and DC-51-W4 (10 μM in lanes22&25; 40 μM in lanes 23&26; 80 μM in lanes 24&27) in 12-well plates for48 hrs (compound-containing fresh media were changed every 24 hrs). Eachcondition was in duplicate. After incubation, insoluble fractions ofcell lysates were collected as described, and analyzed by Westernanalysis with a rabbit anti-human Tau antibody (FIG. 1A), and thenre-probed for α-tubulin as a soluble protein marker and a proteinloading control (FIG. 1B). Lysates of cells grown in the absence oftetracycline (Lanes 1; no TauRD expression) were also analyzed inparallel to show background bands derived from endogenous Tau.

FIG. 2 shows that compound DC-51 and its analogs modulate levels ofTauRD oligomers and monomers in soluble fractions of TREx293-TauRDWT(clone B1) cell cultures as assessed by Western analysis.TREx293-TauRDWT cells were treated with DMSO vehicle control (Lanes 2,15 and 28), compound DC-51 (0.5 μM in lanes 9&12; 1 μM in lanes 10&13;10 μM in lanes 11&14) and its analogs DC-51-W3 (10 μM in lanes 16&19; 40μM in lanes 17&20; 80 μM in lanes 18&21), and DC-51-W4 (10 μM in lanes22&25; 40 μM in lanes 23&26; 80 μM in lanes 24&27) in 12-well plates for48 hrs (compound-containing fresh media were every 24 hrs). Eachcondition was in duplicate. After incubation, soluble fractions of celllysates were collected as described, and analyzed by Western analysiswith a rabbit anti-human Tau antibody (FIG. 2A), and then re-probed forα-tubulin as a soluble protein marker and a protein loading control(FIG. 2B). Lysates of cells grown in the absence of tetracycline (Lane1; no TauRD expression) were also analyzed in parallel to showbackground bands derived from endogenous Tau.

FIGS. 3 a and 3 b show relative levels of soluble/insoluble TauRDoligomers and monomers in compound DC-51-treated TREx293-TauRDWT celllysates by quantitative densitometric analysis of the Western blotsshown in FIGS. 1&2.

FIGS. 4 a and 4 b show relative levels of soluble/insoluble TauRDoligomers and monomers in compound DC-51-W3-treated TREx293-TauRDWT celllysates by quantitative densitometric analysis of the Western blotsshown in FIGS. 1&2.

FIGS. 5 a and 5 b show relative levels of soluble/insoluble TauRDoligomers and monomers in compound DC-51-W4-treated TREx293-TauRDWT celllysates by quantitative densitometric analysis of the Western blotsshown in FIGS. 1&2.

Example 7 Quantitative Analysis of Western Blots

A 50-55% reduction in levels of insoluble TauRD oligomers (30-300 kDa)(FIGS. 1A & 3A), and a 67-83% reduction in levels of insoluble TauRDmonomers (15 kDa) (FIGS. 1A & 3B) was found in cells treated with 0.5-10μM of compound DC-51, when compared to DMSO control (C) (p<0.05-0.01).Treatment with compound DC-51 exhibits no significant effects on levelsof soluble TauRD oligomers, and monomers (FIGS. 2A & 3C-D). The resultsindicate that compound DC-51 is a potential agent that can lowerinsoluble (aggregated) Tau when used at the lower range of micromolarlevels.

A trend of reduction in levels of insoluble TauRD oligomers (30-300 kDa)was found in cells treated with 10-40 μM of compound DC-S 1-W3, whencompared to DMSO control (C) (p>0.05) (FIGS. 1A & 4A). A reduction inlevels of insoluble TauRD monomers (15 kDa) was found in cells treatedwith 10 μM of the compound (p<0.05) (FIGS. 1A & 4B). In addition, a1.54-fold increase in levels of soluble TauRD oligomers, and a 45%reduction in levels of soluble TauRD monomers were found in cellstreated with 10 μM, and 80 μM of the compound, respectively, whencompared to the control (p<0.05)(FIGS. 2A & 4C-D). The results suggestthat compound DC-51-W3 has effects on Tau aggregation under theexperimental conditions.

A trend of increase in levels of insoluble TauRD oligomers (30-300 kDa),and a 65-76% reduction of insoluble TauRD monomers (15 kDa) (p<0.05) wasalso found in cells treated with 40-80 μM of compound DC-5′-W4, whencompared to DMSO control (C) (p>0.05) (FIGS. 1A & 5A-B). A 63-85%reduction in levels of soluble TauRD oligomers, and a 49-95% reductionin levels of soluble TauRD monomers was found in cells treated with40-80 μM, and 10-80 μM of compound DC-5′-W4, respectively, when comparedto DMSO control (C) (p<0.01)(FIGS. 2A & 5C-D). In addition, withtreatment of 40-80 μM of compound DC-51-W4, significant cell toxicitywas also observed, as indicated by reduced cell viability (data notshown), and reduced α-tubulin levels (an indicator of total proteinlevels) (FIG. 2B). The results suggest that compound DC-51-W4 may betoxic when used at >40 μM under the current experimental conditions.

In conclusion, the compounds of this invention modulate tau aggregationand thus potentially are suitable for alleviating tauopathies.

1. A method of disrupting or causing the dissolution of tau aggregatesin a mammal suffering from a tauopathy, comprising administering to themammal suffering from a tauopathy a therapeutically effective amount ofa compound of the formula

or a pharmaceutically acceptable salt thereof where: R is C₅₋₆ alkylenegroup independently substituted with up to two carbonyl groups and up totwo NH groups, and optionally substituted with one or two double bonds,and where R₁ and R₂, and R₃ and R₄ are independently positioned hydroxylgroups.
 2. The method of claim 1, wherein the mammal is a human.
 3. Themethod of claim 1, wherein the amount of the compound administered isbetween 0.1 mg/Kg/day and 1000 mg/Kg/day.
 4. The method of claim 1,wherein the amount of compound administered is between 1 mg/Kg/day and100 mg/Kg/day.
 5. The method of claim 1, wherein the amount of compoundadministered is between 10 mg/Kg/day and 100 mg/Kg/day.
 6. The method ofclaim 1, wherein the tauopathy is selected from the group consisting ofAlzheimer's disease, Pick's disease, progressive supranuclear palsy,corticobasal degeneration, familial frontotemporal dementia/Parkinsonismlinked to chromosome 17, amyotrophic lateralsclerosis/Parkinsonism-dementia complex, argyrophilic grain dementia,dementia pugilistic, diffuse neurofibrillary tangles with calcification,progressive subcortical gliosis and tangle only dementia.
 7. The methodof claim 1 wherein the compound administered is administered by one ofroutes selected from, oral, topical, systemic or parenteral.
 8. A methodof disrupting or causing the dissolution of tau aggregates in a mammalsuffering from a tauopathy, comprising administering to the mammalsuffering from a tauopathy an effective amount of a compound selectedfrom the group consisting of: maleic acid bis(3,4-dihydroxyanilide)(compound DC-0069) and1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenethyl)urea (compoundDC-0078); and pharmaceutically acceptable salts thereof.